转染Survinvin基因的MSCs对小鼠肾脏缺血再灌注损伤的影响

发布时间：2018-02-25 03:01

本文关键词： 骨髓间充质干细胞缺血再灌注损伤 Survinvin 存活能力　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：探讨转染survinvin基因后的骨髓间充质干细胞（MSCs)在肾脏缺血微环境中的存活能力以及对小鼠肾脏缺血再灌注损伤的修复作用能力和机制。方法：复苏、扩增培养已完成空病毒、Survinvin基因转染、鉴定、EGFP标记工作的MSCs，，扩增培养,观察绿色荧光表达情况备用，48只SPF级健康雄性C57BL/6J分为正常对照组12只、IR组（小鼠双侧肾动脉夹闭40min后恢复血供）12只、空病毒转染移植组(MSCs组，小鼠双侧肾动脉夹闭40min后恢复血供24后经尾静脉注射200微升1×106个细胞)12只、survinvin基因转染移植组(SVV/MSCs组，小鼠双侧肾动脉夹闭40min后恢复血供24小时后经尾静脉注射200微升1×106个survinvin基因转染细胞)24只，选取细胞移植后1d、3d、7d、14d不同时间点采集血清行肌酐尿素氮检测，切取小鼠肾脏行石蜡切片观察MSCs存活数目并定量分析，HE染色观察肾组织病理变化及肾小管损害程度评分，ELISA检测肾脏匀浆中细胞因子HGF、bFGF、IL-10的表达情况。结果：复苏、扩增培养出的MSCs增值活力旺盛，荧光状态良好；移植细胞1天后MSCs组与SVV/MSCs组肌酐尿素氮水平明显低于IR对照组（p0.01或p0.05）。肾脏HE染色病理损害评分,干细胞移植组损伤较轻，SVV/MSCs明显低于其他两组（p0.01或p0.05）。第3天、14天存活于肾组织的移植细胞数目SVV/MSCs组远高于MSCs组，表达EGFP的MSCs主要分布于肾小球周围、小血管内壁、肾小管与肾小管之间的间质,而肾小管内壁几乎见不到表达EGFP的MSCs。在肾脏缺血损伤后保护性因子HGF、bFGF、IL-10水平升高SVV/MSCs也比IR组、MSCs组明显的多（P0.01或p0.05)，并且在第14天时与正常对照组已无统计学差异。结论:转染Survinivin基因可以增加MSCs在缺血肾脏中的生存能力，MSCs主要通过其强大的旁分泌作用进一步促进肾脏损伤的修复。
[Abstract]:Aim: to investigate the viability of bone marrow mesenchymal stem cells (MSCs) transfected with survinvin gene in renal ischemic microenvironment and the repair ability and mechanism of renal ischemia-reperfusion injury in mice. Methods: resuscitation, amplification and culture of empty virus Survinvin gene transfection, identification of EGFP labeled MSCs, amplification of culture, To observe the expression of green fluorescence, 48 healthy male C57BL / 6J of SPF grade were divided into 12 normal control group (12 normal control group) (12 normal control group) (12 mice recovered blood supply after bilateral renal artery occlusion for 40 minutes, and 12 mice were treated with empty virus transfection and transplantation group). After bilateral renal artery occlusion for 40 minutes, the blood supply was restored 24 minutes later, and then injected with 200 microliters of 1 脳 106 cells through tail vein. 12 mice were transfected with survinvin gene into SVV / MSCs group. After bilateral renal artery occlusion for 40 minutes, the blood supply was restored 24 hours later, and then injected into tail vein of 24 mice with 1 脳 106 survinvin gene transfection. Serum creatinine urea nitrogen was measured at different time points of 1 day, 3 days, 7 days and 14 days after transplantation. The survival number of MSCs was observed by paraffin section and the pathological changes of renal tissue and the expression of cytokine HGFbFGF- IL-10 in renal homogenate were detected by Elisa. Results: after resuscitation, the proliferative activity and fluorescence state of MSCs were exuberant, the level of creatinine urea nitrogen in MSCs group and SVV/MSCs group was significantly lower than that in IR control group (p0.01 or p0.05), and the pathological damage score of renal HE staining was significantly lower than that in MSCs group and SVV/MSCs group. The number of transplanted cells survived in renal tissue in SVV/MSCs group was much higher than that in MSCs group on the 3rd day after transplantation. The MSCs expressing EGFP was mainly distributed around glomeruli and in the inner wall of small blood vessels, and the number of transplanted cells was significantly lower in the stem cell transplantation group than in the other two groups (P < 0.01 or P 0.05), and the number of the transplanted cells in the renal tissue was significantly higher in the SVV/MSCs group than in the MSCs group. The interstitium between the tubules and the renal tubules, The level of HGFbFGFGF-IL-10 was significantly higher in the renal tubular wall than that in the IR group, and there was no significant difference between them on the 14th day and the control group. Conclusion: transfection of Survinivin gene can increase the viability of MSCs in ischemic kidney and promote the repair of renal injury mainly through its strong paracrine effect.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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